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Immunohistochemical detection of Influenza virus infection in formalin-fixed tissues with anti-H5 monoclonal antibody recognizing FFWTILKP
Journal of Virological Methods 155 (2009) 25–33
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Immunohistochemical detection of Influenza virus infection in formalin-fixed tissues with anti-H5 monoclonal antibody recognizing FFWTILKP Fang He a , Qingyun Du a , YuenFern Ho a , Jimmy Kwang a,b,∗ a b
Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604, Singapore Department of Microbiology, Faculty of Medicine, National University of Singapore, Block MD4, 5 Science Drive 2, Singapore 117597, Singapore
a b s t r a c t Article history: Received 6 May 2008 Received in revised form 8 September 2008 Accepted 15 September 2008 Available online 7 November 2008 Keywords: H5N1 Paraffin sections IHC staining Monoclonal antibody
The worldwide outbreak of avian influenza among poultry species and humans is associated with the H5N1 subtype of avian influenza A virus (AIV). This highlighted the need to develop safe H5 AIV diagnostic methods. 7H10, an H5-specific monoclonal antibody (Mab), can be used for immunohistochemical (IHC) staining for formalin-fixed tissue. An assortment of H5N1 tissue specimens infected naturally in paraffin sections from Asia, between years 2002–2006, including one human specimen, were tested. 7H10 detected H5 infection in all of these tissue samples infected naturally. In addition, 24 different human H5N1 isolates from Indonesia, 5 avian H5 isolates and 3 non-H5 isolates from Asia were inoculated into BALB/C mice and chicken embryos. Among these influenza viruses, 7H10 detected 28 of the 29 H5 virus strains by immunohistochemical staining, while none of non-H5 strains used in this study could be detected by 7H10, confirming its specificity to H5. Further, the eight-residue-long linear epitope, “FFWTILKP”, identified through epitope mapping, enables 7H10 to detect >98.3% of H5 subtype viruses reported worldwide before 2007. This study describes a specific H5 diagnostic system with minimal possibility of exposure to live virus based on immunochemical staining. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Highly pathogenic H5N1 avian influenza has caused global concerns for public health and continues to spread throughout the world (Peiris et al., 2007). Current areas affected include essentially all of Asia, Europe and to a lesser extent northern and western Africa (Apisarnthanarak et al., 2007; Babakir-Mina et al., 2007; Park and Glass, 2007). Continuous occurrences of H5N1 infection in humans were reported in Indonesia (Sedyaningsih et al., 2007). The current situation in Indonesia of H5N1 infection provides an extensive reservoir of H5 subtype influenza viruses that could infect humans. Therefore, a safe H5 diagnostic system, without accidental release or exposure to live virus, is required. Immunohistochemical (IHC) staining is an effective method for viral detection based on its ability to locate target antigens in tissue sections (Nuovo, 2006). It provides qualitative information about the severity of infection. In particular, IHC staining with paraffin sections enables detection of viral antigens in formalin-fixed tissues, in which viral pathogens have been inactivated, to reduce
the risk of accidental release or exposure to live virus. To date, IHC staining was used to detect or characterize influenza viral infection with antibodies targeting nucleoprotein or hemagglutinin (Gao et al., 1999; Gu et al., 2007; Guarner et al., 2006, 2000; To et al., 2001). However, there are no reports about any specific detection of H5N1 virus in paraffin-embedded tissues by IHC staining, possibly due to the lack of effective H5-specific Mabs targeting epitopes retrieved in formalin-fixed tissues. Linear epitopes on the antigen are independent of protein conformation. This property allows a linear epitope to be recognized by antibodies after fixation or denaturing treatments, which provides the potential to produce an H5-specific Mab used in formalin-fixed tissue sections (Koch and Csonka, 1958). In this study a method was developed for the immunohistochemical detection of H5 AIV in paraffin-embedded tissue sections with an H5-specific monoclonal antibody. This method successfully detects H5 infection in tissues collected from a variety of avian and mammalian species, based on an H5-specific and common epitope.
2. Methods ∗ Corresponding author at: Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604, Singapore. Tel.: +65 6872 7473; fax: +65 6872 7007. E-mail address: [email protected] (J. Kwang). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.09.016
2.1. Viruses and tissues All 24 H5N1 human isolates and one H5N1 infected human lung tissue were obtained from National Institute of Health, Research
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and Development, Indonesia. Two H5N1 avian strains and 10 paraffin-embedded avian tissue blocks were from Institute of Pertanian in Bogor, Indonesia. Another 9 avian tissue paraffin blocks were from Hong Kong’s Tai Lung Veterinary Laboratory. VNH5N1PR8/CDC-RG, a non-pathogenic reassortant AIV strain containing the HA and NA genes of strain A/Vietnam/1203/04, and H1N1 (A/Puerto Rico/8/34) were obtained from the Atlanta branch of the Center for Disease Control. Non-H5N1 AIV strains, H5N2, H5N3, H3N2, H7N1 and H9N2, were obtained from the AgriFood and Veterinary Authority (AVA) of Singapore. Besides, H2N9 (A/Duck/Nanchang/4-184/00), H6N8 (A/shorebird/DE/12/04) and one H5N1 (A/turkey/Turkey/1/05) were generated by reverse genetics according to standard protocols. 11-day-old chicken embryos were inoculated with 10 ELD50 AIV, and mice were intranasally infected with 200 ELD50 AIV. Embryos were dissected at 24 h post-inoculation and mice were euthanized 5 days post-infection. Organs, such as lungs and brains, were immediately fixed in 10% buffered formalin. All experiments with high pathogenic viruses (Tanaka et al., 2003) were performed in a biosafety level 3 containment laboratory. 2.2. H5 monoclonal antibodies Attenuated strain VNH5N1-PR8/CDC-RG was used to immunize 6 to 8-week-old female BALB/c mice with two doses of 20–60 g antigen over two weeks. Mice having the highest antibody titers were used for splenocyte recovery and fusion to SP2/0 myeloma cells, as described previously (Ross et al., 2000; Yokoyama et al., 2006). Antibodies were isotyped using a mouse Mab isotyping kit (Amersham Biosciences). Hemagglutination inhibition (HI) tests were carried out in microtiter plates with 1% suspension of chicken red blood cells. For neutralization tests, 2 × 104 ml−1 of MDCK cells were allowed to grow to 70–90% of confluence. Allantoic fluids with H5N1 were 10 fold serially diluted from 10−1 to 10−8 , and tested for TCID50 . Using Reed and Muench mathematical technique, the infectivity titer was expressed as TCID50 /100 l and the viruses were diluted to 100 TCID50 in 50 l. Subsequently, 100 TCID50 viruses were incubated with antibody samples for 1 h at 37 ◦ C and inoculated into MDCK cells. The cells were then incubated at 37 ◦ C and CPE was observed at 96 h post-infection. Besides, a cell-based ELISA was performed to determine neutralization titer according to standard procedures. 2.3. Immunofluorescence assays Madin Darby canine kidney (MDCK) cells were infected with AIVs at 37 ◦ C with 5% CO2 . Spodoptera frugiperda 9 (SF9) cells were infected with recombinant baculovirus expressing HA. The infected cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were incubated with 7H10 for 1 h at 37 ◦ C, washed and then incubated with 1:100 diluted fluorescein isothiocyanate
(FITC)-conjugated anti-mouse antibody (DAKO Cytomation) for 1 h at 37 ◦ C. Samples were observed by fluorescence microscopy. 2.4. Western blot Recombinant HA1 protein (A/goose/Guangdong/97/H5N1) (Nwe et al., 2006) was subjected to 12% SDS-PAGE and transferred on nitrocellulose membranes. Blocked membranes were incubated with 7H10, washed and treated with 1:2000 diluted HRP anti-mouse antibody (DAKO Cytomation). Signals were developed with 3,3 -diaminobenzidine (DAB). For epitope mapping, truncated or mutated fragments were amplified from full-length HA1 (A/goose/Guangdong/97/H5N1) and expressed in pQE vector (Qiagen). The truncated or mutated proteins were screened in immunoblotting with 7H10. 2.5. Immunohistochemical staining Tissue specimens were fixed in formalin, embedded in paraffin blocks cut at 2–4 m (Leica Autocut microtome 2055) and attached to poly-l-lysine coated glass slides. Slides were de-paraffinized using Histo-choice (Amersco) and rehydrated in ethanol baths graduated sequentially (Evensen et al., 1994; Fitzgerald et al., 1995; Hamir et al., 1993; Larochelle and Magar, 1995). For antigen retrieval, sections were pressurized in 10 mM citric acid (pH 6) at 120 ◦ C for 5 min as described previously (Dilbeck and McElwain, 1994). Slides were then treated with 3% H2 O2 for 20 min, blocked in 1% nonfat milk (BioRad) for 30 min, and incubated with Mabs for 1 h and with HRP anti-mouse for 30 min at room temperature. Finally, the slides were developed with AEC+ chromogenic substrate (Dako Cytomation) for 10 min, stained with Lillie-Mayer hematoxylin for 2 min and mounted using aqueous mounting media. 3. Results 3.1. Characterization of monoclonal antibody 7H10 The isotype of Mab 7H10 is IgG1. With 7H10, fluorescence signal was observed in MDCK cells inoculated with H5N1 [(A/turkey/Turkey/1/05), (A/Vietnam/1203PR8/H5N1), (A/ Indonesia/TLL101/H5N1) and (A/Indonesia/CDC594/H5N1)], H5N2 (A/Chicken/Singapore/98) and H5N3 (A/Chicken/Singapore/97), while no significant signal was detected in other non-H5 infected cells [(A/Puerto Rico/8/34/H1N1), (A/Duck/Nanchang/4184/00/H2N9), (A/chicken/Singapore/02/H3N2), (A/shorebird/DE/ 12/04/H6N8) and (A/chicken/Singapore/94/H7N1)] or uninfected cells (Fig. 1A). This result validates that 7H10 can recognize H5 subtype AIV specifically. Also, the same experiment was performed with recombinant baculovirus-infected SF9 cells (Fig. 1A). HA expressed in recombinant baculovirus was cloned from an H5 strain (A/goose/Guangdong/97/H5N1). Fluorescence signal was observed
Table 1 Lack of neutralizing activity of 7H10 to H5 AIVs. Monoclonal antibodies
7H10 5B5 8B6c 3F1d a b c d
A/Vietnam/1203/04 (clade 1)
A/Indonesia/CDC594/06 (clade 2.1)
HIa
VNb
HI
VN
1 1 32 1
10 10 640 10
1 1 16 1
10 10 320 10
HI: Titer in hemagglutination inhibition; titer of 1 indicates a negative result. VN: Titer in virus neutralization with MDCK cells; titer of 10 indicates a negative result. 8B6 is a neutralization antibody to H5, used as a positive control. 3F1 is a non-HA antibody, used as a negative control.
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Fig. 1. Characterization of the monoclonal antibody 7H10. (A) Recognition of HA in H5 AIV-infected MDCK cells and baculovirus expressed rHA1 by 7H10 in immunofluorescence assays. MDCK and SF9 cells were infected with variant subtypes AIV or recombinant baculoviruses, respectively; H5N1*: MDCK cells were infected with H5N1 human strain (A/Indonesia/CDC594/2006); the rest were infected with avian strains or attenuated strains from countries as indicated, which are A/turkey/Turkey/1/05, A/Vietnam/1203PR8/H5N1, A/Indonesia/TLL101/H5N1, A/chicken/Singapore/98/H5N2, A/chicken/Singapore/97/H5N3, A/Puerto Rico/8/34/H1N1, A/Duck/Nanchang/4-184/00/H2N9, A/chicken/Singapore/02/H3N2, A/shorebird/DE/12/04/H6N8 and A/chicken/Singapore/94/H7N1. (B) Western blot. 7H10 can detect HA1 protein of H5N1 virus (A/goose/Guangdong/97 (H5N1)) expressed in E. coli total cell lysate. Negative control is the blotting with RPMI medium.
only in H5-expressing SF9 cells, indicating that 7H10 recognizes the H5 protein, but not any other viral proteins from H5 AIV. Further, in Western blot, recombinant E. coli-expressed H5 HA1 protein (A/goose/Guangdong/97/H5N1) was detected from total cell lysate with 7H10. The protein size shown on the nitrocellulose membrane was 36 kDa (Fig. 1B), which is equivalent to the molecular weight of HA1. This finding reveals that 7H10 reacts against a linear epitope on the HA1 protein of H5. Meanwhile, 7H10 fails to neutralize any of H5 AIVs either in MDCK cells or in chicken embryos. It does not have HI activity to any of H5 AIVs tested (Table 1), confirming that 7H10 does not target any neutralizing epitope. 3.2. Detection of natural infection of H5 subtype virus in formalin-fixed tissues H5N1 AIV-infected tissue samples from Hong Kong, which were collected from the years 2002 to 2006, were tested with 7H10 in immunohistochemical staining. These tissues include different types of organs, such as brain, kidney, liver, lung and pancreas from various avian species, including magpie robin (Copsychus saularis), pond heron (Ardeola bacchus), chicken (Gallus gallus) and grey heron (Ardea cinerea). H5 expression was observed in every infected sample in the staining with 7H10 (Fig. 2). In kidneys, there was a wide distribution of intensive signals throughout the renal tubular cells.
In the lung tissue, the bronchiole lumen showed strong signals at the lining of the epithelial cells. Also, H5 expression was found in individual epithelia. In the brain tissue, the staining signal was observed in cytoplasm of glial cells and neurons. None of samples stained with RPMI medium displayed any specific signal. These data indicate that 7H10 recognizes H5 expression in formalin-fixed tissues. Further, in avian tissue specimens infected with H5 AIV from Indonesia, similar positive results were obtained by 7H10. These samples include different organs, such as thymus, kidney, liver, lung, ovary and pancreas from 2005 to 2006. This underlines that 7H10 can detect the expression of H5 strains, from different regions, in formalin-fixed tissues. 3.3. Detection of experimental infection with variant H5 AIV human isolates in formalin-fixed tissues To investigate further whether the 7H10 is able to detect variant H5 AIVs, especially H5N1 human isolates, 24 different human H5N1 strains from Indonesia and 8 avian strains were collected from Indonesia, Vietnam and Singapore, including 3 non-H5 strains. Experimental infection was performed in chicken embryos and mice with these viruses. Brain tissues from infected chicken embryos were tested with 7H10 to locate H5 antigen expression.
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Fig. 2. Immunohistochemical detection of H5N1 AIV in paraffin-embedded tissue sections of different organs from avian species infected with Hong Kong strains using 7H10. Organs obtained from magpie robin (C. saularis) were preserved in paraffin embedding (brain, lung and kidney) and subjected to IHC staining as described. (−): negative controls stained with RPMI medium. Magnification, ×32.
H5 expression was observed exclusively in cytoplasm of glial cells and neurons from infected chicken embryos (Fig. 3A). Infected cells were extensively distributed through the entire brain tissue. Compared with normal brain tissues, glial cell proliferation was also observed among H5-expressing cells as lesions caused by highly pathogenic AIV infection. Among all, 23 of the 24 human strains and all of these avian H5 strains were successfully detected by 7H10 in IHC staining, while none of those samples infected with non-H5 strains presented any positive staining with 7H10 (Fig. 3C). The antibody targeting nucleoprotein was used to validate AIV infection in these chicken embryos. These results confirm that 7H10 is an H5-
specific monoclonal antibody without cross activity to any other AIV subtypes in IHC staining in formalin-fixed tissues. Mice infected experimentally with human H5N1 isolates were used in this study to test 7H10 activity in mammalian tissues. Among the mouse organs tested, positive signals were mainly observed in lung and brain tissues. In the infected lung tissue, the H5 viral antigen expresses in cytoplasm of the respiratory epithelium and alveolar macrophages (Fig. 4A and B), as well as in cellular debris of the bronchi lumen and bronchioles. In the infected brain tissues, H5 antigen expression was observed in cytoplasm of individual neurons and glial cells, and parts of infected cells became
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Fig. 3. Immunohistochemical detection of H5N1 AIV in paraffin-embedded brain tissue sections of chicken embryos infected experimentally with an Indonesia human strain (A/Indonesia/TLL001/2006/H5N1) using 7H10. (A) Viral antigens were detected in the brain tissue of 10-day-old chicken embryos. Arrow: glial cell proliferation caused by infection; (B) no viral antigen and lesion was detected in the brain tissue of uninfected chicken embryos stained with 7H10 in IHC assays; (C) brain tissues from chicken embryos infected with variant AIV strains were stained with 7H10 and anti-NP antibody in IHC assays. H5N1: A/Indonesia/TLL005/06/H5N1H9N2; H3N2: A/chicken/Singapore/02/H3N2; H7N1: A/chicken/Singapore/94/H7N1; A/chicken/Singapore/98/H9N2. Magnification, ×96.
degenerative (Fig. 4C and D), indicating systemic infection of these highly pathogenic H5N1 strains. This result reflects that 7H10, which is able to detect H5 in formalin-fixed tissue, is effective in mammalian tissues as well as in avian tissues. 3.4. Immunohistochemical detection of H5N1 infection in one human tissue sample with 7H10 A human lung tissue was collected from one patient who died from H5N1 AIV infection in 2006. H5N1 infection in this case was identified by reverse-transcription PCR performed previously in Indonesia. The human lung specimen was fixed in 10% buffered formalin and tested with 7H10 in IHC staining. H5 expression due to H5N1 infection was detected in lung epithelium cells (Fig. 5A). Compared to mouse specimens, H5 signals were sporadic in the human lung tissue. In those infected cells, H5 expression distributed throughout cytoplasm. This result indicates that 7H10based IHC method is sensitive enough to detect H5 infection in human tissues, suggesting that 7H10 can be employed for additional studies on human pathology during H5N1 infection. 3.5. Identification of an H5-specific linear epitope for 7H10 Immunoblotting using 7H10 showed that the epitope was primarily found in the overlapping region of fragment B and C (aa 201–266) from the HA1 of H5 subtype (A/goose/Guangdong/97/ H5N1). To identify the C terminal of this epitope, 8 truncated
fragments were designed and screened with 7H10 (Fig. 6A) in immunoblotting. The amino acid 251 on HA1 was found to be the last amino acid of the epitope for 7H10. To locate the N terminal of this epitope, 8 mutated fragments were designed by primers with mutated sequences and screened with 7H10. Among the 8 mutants, amino acids 240–247 on HA1 were changed to alanine, individually, by specific primers. As shown in Western blot, the first amino acid in the epitope is the amino acid 244 on HA1 (Fig. 6B). This indicates that the linear epitope of 7H10 is exactly located from the amino acid 244 to 251 on the hemagglutinin of H5 subtype, the sequence of which is “Phe Phe Trp Thr Ile Leu Lys Pro” (FFWTILKP). To verify the epitope sequence further, HA gene-sequencing was performed with the 24 human AIV strains. 21 of them carry identical epitope sequences, while two detectable strains (A/Indonesia/CDC329/2006, ABI36050 and A/Indonesia/CDC326/ 2006, ABI36048) have the compatible substitution in one residue on the epitope (FFWTILNP), suggesting the detection range of 7H10 for H5 strains could be even larger. However, for the only strain which can not be detected in our immunohistochemistry test (A/Indonesia/CDC669/2006, ABI36450), the epitope was found to be “FFWAILKP”. The mutation from threonine to alanine corresponds to a mutant designed for epitope mapping, which could not be detected by 7H10 in immunoblotting either. The detection range of 7H10 for H5 subtype AIV was further studied by searching the epitope sequence of 7H10 in the database of AIV genome provided by NCBI influenza resource (Table 2). Up to June of 2007, among 1088 full-length sequences of H5 in the
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Fig. 4. Immunohistochemical detection of Indonesia H5N1 AIV in paraffin-embedded lung and brain tissue sections of mice infected experimentally with an Indonesia human strain (A/Indonesia/TLL011/2006/H5N1) using 7H10. (A), (B) Viral antigen expression in respiratory epithelial cells or alveolar macrophage. (C), (D) Viral antigen expression in neurons and glial cells. (A), (C), magnification: ×32; (B), (D), magnification: ×96.
database, 972 cases contain the identical epitope sequence and 98 cases contain compatible epitope sequence for detection, indicating that 7H10 is able to detect over 98.3% of H5 strains reported. Besides, the detection range of 7H10 reaches 100% for H5 AIV strains
from Hong Kong and Vietnam. 7H10 can detect 93.58% of H5 AIV strains from Indonesia due to 6 cases with the undetectable epitope “FFWAILKP”. Meanwhile, no sequence from any other hemagglutinin subtype was found to contain any detectable epitope for 7H10
Fig. 5. Immunohistochemical detection of H5N1 (A/Indonesia/TLL012/2006/H5N1) infection in paraffin-embedded lung tissue sections of humans. (A) Expression of H5 antigen in respiratory epithelial cells; (B) negative controls stained with RPMI medium. Magnification, ×96.
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Fig. 6. Mapping of the epitope of 7H10. (A) Schematic diagram of the hemagglutinin protein 1, showing the expression constructs of the HA1 fragments and their interaction with 7H10 in Western blot. aa, amino acid. (B) Schematic diagram of the mutated hemagglutinin 1 fragments, showing the constructs for the expression of the different mutations on the HA1 fragments and their interaction with 7H10. “+”: positive by 7H10 in Western blot. “−”: negative by 7H10 in Western blot.
Biosafety Level 3 facilities, there is an urgent need for safe and effective diagnostic methods among developing countries that are plagued from H5N1 AIV. Under normal circumstances, viral virus infectivity in the infected tissues is inactivated by the fixation process, which might remove the lipid envelop and destroy natural conformation of viral hemagglutinin (King, 1991; Koch and Csonka, 1958; Okamoto et al., 2004). Therefore, working on formalin-fixed tissues, the IHC method described in this study provides a stepping stone for regulatory agencies to a
in NCBI database, confirming “FFWTILKP” is an H5-specific and common epitope. 4. Discussion Immunohistochemical detection of H5 infection is important for a better understanding in the pathology of H5 AIV in both birds and humans (Gu et al., 2007; Lee et al., 2005; Uiprasertkul, 2007; Uiprasertkul et al., 2005). Due to the lack of sufficient Table 2 H5-specific linear epitopes of 7H10 among H5 AIVs in database by June 2007. Subtype
Region
Sequences with identical epitope
Sequences with detectable epitope
Total sequences
Detectable range
H5 H5 H5 H5 H5 H1 H3 H6 H7 H9
Any Asia Hong Kong Vietnam Indonesia Any Any Any Any Any
972 753 122 127 88 0 0 0 0 0
1070 784 122 129 102 0 0 0 0 0
1088 800 122 129 109 599 1749 204 238 184
98.35% 98% 100% 100% 93.58% 0 0 0 0 0
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Fig. 7. A new H5 monoclonal antibody was used in H5 detection by IHC staining. Brain tissue sections of chicken embryos infected experimentally with an Indonesia human strain (A/Indonesia/CDC669/H5N1) were stained using the new Mab 5B5 or 7H10. Positive signals were observed in 669-infected cells by 5B5.
safer diagnosis on H5 infected animal tissues (Wootton et al., 2006). The results in this report indicate that 7H10 enables effective detection of H5 antigen to be performed in formalin-fixed tissue sections. This finding, together with its activity in Western blotting and the lack of HI and neutralization activities, indicates that 7H10 recognizes a linear epitope on the HA protein. Since a linear epitope is independent of conformation, recognition by the certain Mab is simply determined by the amino acid sequence of the epitope. By Sequence Polymorphism analysis (Biohealth base), each of residues on 7H10 epitope is identified as the major residue among H5 subtype. Even if some variants do not contain the exact epitope sequence, substitutions with similar amino acids are compatible for the antibody binding. Therefore, more than 98.3% of H5N1 AIV worldwide, could be detected by 7H10 based on the exact or compatible epitope. Since such dominant sequence is not found in any other subtype, the sequence “FFWTILKP” is identified as a common and specific epitope on the H5 subtype hemagglutinins. Among those H5 strains with undetectable epitope sequence, 6 strains derived from H5N1 cases reported in 2006 in Indonesia, which carry the “FFWAILKP” epitope. This phenomenon leads to concerns about the vulnerability of 7H10-based detection due to the possibility of antigenic drifts. Therefore, another anti-H5 monoclonal antibody, termed as 5B5, was generated with the antigen from an Indonesia H5N1 strain (A/Indonesia/CDC669/2006), which cannot be detected by 7H10 (Fig. 7). 5B5 recognizes a linear epitope existing in all Indonesia H5 AIV strains and most of non-Indonesia H5 strains (Table 1). Furthermore, the combination of the two Mabs can detect 100% of H5 AIV strains in Indonesia, Hong Kong and Vietnam. Future work will focus on the updating of this Mab cocktail in H5 AIV diagnosis. In addition, all of H5 strains reported in Genebank in 2007 from Indonesia contain the exact “FFWTILKP” epitope, indicating that this epitope continues to be dominant and conserved in Indonesia at current time. The difference in viral antigen distribution and intensity among different species reflects the difference in pathogenicity of H5N1 virus among various host species. This is mainly caused by the difference in the distribution of sialic acid receptor for AIVs (Szollosy
et al., 1952). As shown in the results, the method with 7H10 is able to detect H5 AIV infection in tissue sections from mammals, including humans, indicating its sensitivity in detection and its potential in promoting the understanding of H5 viral pathology in human infection. In conclusion, the use of 7H10, in diagnostic IHC staining, presents three advantages over the current methodologies. Firstly, working on formalin-fixed tissue, 7H10 provides a safe diagnostic approach in H5N1-AIV studies. This approach realizes safer detection of H5 AIV in suspicious samples as they will neither infect humans nor contaminate the environment. Secondly, 7H10 could identify the highly pathogenic H5 subtype AIV efficiently and specifically, which is effective in almost all of H5 influenza viruses in the database without cross activity to other subtypes. This could mean a breakthrough in the AIV diagnosis in the light of its specific detection of H5 viral antigen in formalin-fixed tissue sections. Lastly, the ability of 7H10 to accurately locate H5 viral antigen in infected tissue, facilitates microbiologists and pathologists to obtain information about the stage and pathology of AIV infection in tissue samples in the studies on H5 AIV. Acknowledgements We thank the National Institute of Health, Research and Development, Indonesia, Hong Kong Agriculture and Fisheries Department Tai Lung Veterinary Lab and Institute of Pertanian in Bogor, Indonesia, for contributing H5N1 infected specimens or clinical samples, Ms. Yu Zhu for tissue sectioning, Ms. Huiting Ho for providing virus samples by reverse genetics and Mr. Hongliang Qian for providing control Mabs. References Apisarnthanarak, A., Warren, D.K., Fraser, V.J., 2007. Issues relevant to the adoption and modification of hospital infection-control recommendations for avian influenza (H5N1 infection) in developing countries. Clin. Infect. Dis. 45, 1338–1342. Babakir-Mina, M., Balestra, E., Perno, C.F., Aquaro, S., 2007. Influenza virus A (H5N1): a pandemic risk? New Microbiol. 30, 65–78.
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Nwe, N., He, Q., Damrongwatanapokin, S., Du, Q., Manopo, I., Limlamthong, Y., Fenner, B.J., Spencer, L., Kwang, J., 2006. Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture. BMC Microbiol. 6, 16. Okamoto, S., Kawabata, S., Fujitaka, H., Uehira, T., Okuno, Y., Hamada, S., 2004. Vaccination with formalin-inactivated influenza vaccine protects mice against lethal influenza Streptococcus pyogenes superinfection. Vaccine 22, 2887– 2893. Park, A.W., Glass, K., 2007. Dynamic patterns of avian and human influenza in east and southeast Asia. Lancet Infect. Dis. 7, 543–548. Peiris, J.S., de Jong, M.D., Guan, Y., 2007. Avian influenza virus (H5N1): a threat to human health. Clin. Microbiol. Rev. 20, 243–267. Ross, T.M., Xu, Y., Bright, R.A., Robinson, H.L., 2000. C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge. Nat. Immunol. 1, 127–131. Sedyaningsih, E.R., Isfandari, S., Setiawaty, V., Rifati, L., Harun, S., Purba, W., Imari, S., Giriputra, S., Blair, P.J., Putnam, S.D., Uyeki, T.M., Soendoro, T., 2007. Epidemiology of cases of H5N1 virus infection in Indonesia, July 2005–June 2006. J. Infect. Dis. 196, 522–527. Szollosy, E., Invanovics, G., Horvath, S., 1952. Distribution of the receptor substance of influenza and related viruses in tissue elements of different animal species. II. The virus absorbing capacity of the respiratory tract of various vertebrates. Acta Physiol. Hung. 3, 431–440. Tanaka, H., Park, C.H., Ninomiya, A., Ozaki, H., Takada, A., Umemura, T., Kida, H., 2003. Neurotropism of the 1997 Hong Kong H5N1 influenza virus in mice. Vet. Microbiol. 95, 1–13. To, K.F., Chan, P.K., Chan, K.F., Lee, W.K., Lam, W.Y., Wong, K.F., Tang, N.L., Tsang, D.N., Sung, R.Y., Buckley, T.A., Tam, J.S., Cheng, A.F., 2001. Pathology of fatal human infection associated with avian influenza A H5N1 virus. J. Med. Virol. 63, 242–246. Uiprasertkul, M., 2007. Apoptosis and pathogenesis of avian influenza A (H5N1) virus in humans. Emerg. Infect. Dis. 13, 708–712. Uiprasertkul, M., Puthavathana, P., Sangsiriwut, K., Pooruk, P., Srisook, K., Peiris, M., Nicholls, J.M., Chokephaibulkit, K., Vanprapar, N., Auewarakul, P., 2005. Influenza A H5N1 replication sites in humans. Emerg. Infect. Dis. 11, 1036– 1041. Wootton, S.H., Scheifele, D.W., Mak, A., Petric, M., Skowronski, D.M., 2006. Detection of human influenza virus in the stool of children. Pediatr. Infect. Dis. J. 25, 1194–1195. Yokoyama, M.W., Christensen, M., Santos, G.D. and Miller, D., 2006. Production of monoclonal antibodies. Curr. Protoc. Immunol. Chapter 2, Unit 2 5.
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Immunohistochemical detection of Influenza virus infection in formalin-fixed tissues with anti-H5 monoclonal antibody recognizing FFWTILKP Fang He a , Qingyun Du a , YuenFern Ho a , Jimmy Kwang a,b,∗ a b
Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604, Singapore Department of Microbiology, Faculty of Medicine, National University of Singapore, Block MD4, 5 Science Drive 2, Singapore 117597, Singapore
a b s t r a c t Article history: Received 6 May 2008 Received in revised form 8 September 2008 Accepted 15 September 2008 Available online 7 November 2008 Keywords: H5N1 Paraffin sections IHC staining Monoclonal antibody
The worldwide outbreak of avian influenza among poultry species and humans is associated with the H5N1 subtype of avian influenza A virus (AIV). This highlighted the need to develop safe H5 AIV diagnostic methods. 7H10, an H5-specific monoclonal antibody (Mab), can be used for immunohistochemical (IHC) staining for formalin-fixed tissue. An assortment of H5N1 tissue specimens infected naturally in paraffin sections from Asia, between years 2002–2006, including one human specimen, were tested. 7H10 detected H5 infection in all of these tissue samples infected naturally. In addition, 24 different human H5N1 isolates from Indonesia, 5 avian H5 isolates and 3 non-H5 isolates from Asia were inoculated into BALB/C mice and chicken embryos. Among these influenza viruses, 7H10 detected 28 of the 29 H5 virus strains by immunohistochemical staining, while none of non-H5 strains used in this study could be detected by 7H10, confirming its specificity to H5. Further, the eight-residue-long linear epitope, “FFWTILKP”, identified through epitope mapping, enables 7H10 to detect >98.3% of H5 subtype viruses reported worldwide before 2007. This study describes a specific H5 diagnostic system with minimal possibility of exposure to live virus based on immunochemical staining. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Highly pathogenic H5N1 avian influenza has caused global concerns for public health and continues to spread throughout the world (Peiris et al., 2007). Current areas affected include essentially all of Asia, Europe and to a lesser extent northern and western Africa (Apisarnthanarak et al., 2007; Babakir-Mina et al., 2007; Park and Glass, 2007). Continuous occurrences of H5N1 infection in humans were reported in Indonesia (Sedyaningsih et al., 2007). The current situation in Indonesia of H5N1 infection provides an extensive reservoir of H5 subtype influenza viruses that could infect humans. Therefore, a safe H5 diagnostic system, without accidental release or exposure to live virus, is required. Immunohistochemical (IHC) staining is an effective method for viral detection based on its ability to locate target antigens in tissue sections (Nuovo, 2006). It provides qualitative information about the severity of infection. In particular, IHC staining with paraffin sections enables detection of viral antigens in formalin-fixed tissues, in which viral pathogens have been inactivated, to reduce
the risk of accidental release or exposure to live virus. To date, IHC staining was used to detect or characterize influenza viral infection with antibodies targeting nucleoprotein or hemagglutinin (Gao et al., 1999; Gu et al., 2007; Guarner et al., 2006, 2000; To et al., 2001). However, there are no reports about any specific detection of H5N1 virus in paraffin-embedded tissues by IHC staining, possibly due to the lack of effective H5-specific Mabs targeting epitopes retrieved in formalin-fixed tissues. Linear epitopes on the antigen are independent of protein conformation. This property allows a linear epitope to be recognized by antibodies after fixation or denaturing treatments, which provides the potential to produce an H5-specific Mab used in formalin-fixed tissue sections (Koch and Csonka, 1958). In this study a method was developed for the immunohistochemical detection of H5 AIV in paraffin-embedded tissue sections with an H5-specific monoclonal antibody. This method successfully detects H5 infection in tissues collected from a variety of avian and mammalian species, based on an H5-specific and common epitope.
2. Methods ∗ Corresponding author at: Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604, Singapore. Tel.: +65 6872 7473; fax: +65 6872 7007. E-mail address: [email protected] (J. Kwang). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.09.016
2.1. Viruses and tissues All 24 H5N1 human isolates and one H5N1 infected human lung tissue were obtained from National Institute of Health, Research
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and Development, Indonesia. Two H5N1 avian strains and 10 paraffin-embedded avian tissue blocks were from Institute of Pertanian in Bogor, Indonesia. Another 9 avian tissue paraffin blocks were from Hong Kong’s Tai Lung Veterinary Laboratory. VNH5N1PR8/CDC-RG, a non-pathogenic reassortant AIV strain containing the HA and NA genes of strain A/Vietnam/1203/04, and H1N1 (A/Puerto Rico/8/34) were obtained from the Atlanta branch of the Center for Disease Control. Non-H5N1 AIV strains, H5N2, H5N3, H3N2, H7N1 and H9N2, were obtained from the AgriFood and Veterinary Authority (AVA) of Singapore. Besides, H2N9 (A/Duck/Nanchang/4-184/00), H6N8 (A/shorebird/DE/12/04) and one H5N1 (A/turkey/Turkey/1/05) were generated by reverse genetics according to standard protocols. 11-day-old chicken embryos were inoculated with 10 ELD50 AIV, and mice were intranasally infected with 200 ELD50 AIV. Embryos were dissected at 24 h post-inoculation and mice were euthanized 5 days post-infection. Organs, such as lungs and brains, were immediately fixed in 10% buffered formalin. All experiments with high pathogenic viruses (Tanaka et al., 2003) were performed in a biosafety level 3 containment laboratory. 2.2. H5 monoclonal antibodies Attenuated strain VNH5N1-PR8/CDC-RG was used to immunize 6 to 8-week-old female BALB/c mice with two doses of 20–60 g antigen over two weeks. Mice having the highest antibody titers were used for splenocyte recovery and fusion to SP2/0 myeloma cells, as described previously (Ross et al., 2000; Yokoyama et al., 2006). Antibodies were isotyped using a mouse Mab isotyping kit (Amersham Biosciences). Hemagglutination inhibition (HI) tests were carried out in microtiter plates with 1% suspension of chicken red blood cells. For neutralization tests, 2 × 104 ml−1 of MDCK cells were allowed to grow to 70–90% of confluence. Allantoic fluids with H5N1 were 10 fold serially diluted from 10−1 to 10−8 , and tested for TCID50 . Using Reed and Muench mathematical technique, the infectivity titer was expressed as TCID50 /100 l and the viruses were diluted to 100 TCID50 in 50 l. Subsequently, 100 TCID50 viruses were incubated with antibody samples for 1 h at 37 ◦ C and inoculated into MDCK cells. The cells were then incubated at 37 ◦ C and CPE was observed at 96 h post-infection. Besides, a cell-based ELISA was performed to determine neutralization titer according to standard procedures. 2.3. Immunofluorescence assays Madin Darby canine kidney (MDCK) cells were infected with AIVs at 37 ◦ C with 5% CO2 . Spodoptera frugiperda 9 (SF9) cells were infected with recombinant baculovirus expressing HA. The infected cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were incubated with 7H10 for 1 h at 37 ◦ C, washed and then incubated with 1:100 diluted fluorescein isothiocyanate
(FITC)-conjugated anti-mouse antibody (DAKO Cytomation) for 1 h at 37 ◦ C. Samples were observed by fluorescence microscopy. 2.4. Western blot Recombinant HA1 protein (A/goose/Guangdong/97/H5N1) (Nwe et al., 2006) was subjected to 12% SDS-PAGE and transferred on nitrocellulose membranes. Blocked membranes were incubated with 7H10, washed and treated with 1:2000 diluted HRP anti-mouse antibody (DAKO Cytomation). Signals were developed with 3,3 -diaminobenzidine (DAB). For epitope mapping, truncated or mutated fragments were amplified from full-length HA1 (A/goose/Guangdong/97/H5N1) and expressed in pQE vector (Qiagen). The truncated or mutated proteins were screened in immunoblotting with 7H10. 2.5. Immunohistochemical staining Tissue specimens were fixed in formalin, embedded in paraffin blocks cut at 2–4 m (Leica Autocut microtome 2055) and attached to poly-l-lysine coated glass slides. Slides were de-paraffinized using Histo-choice (Amersco) and rehydrated in ethanol baths graduated sequentially (Evensen et al., 1994; Fitzgerald et al., 1995; Hamir et al., 1993; Larochelle and Magar, 1995). For antigen retrieval, sections were pressurized in 10 mM citric acid (pH 6) at 120 ◦ C for 5 min as described previously (Dilbeck and McElwain, 1994). Slides were then treated with 3% H2 O2 for 20 min, blocked in 1% nonfat milk (BioRad) for 30 min, and incubated with Mabs for 1 h and with HRP anti-mouse for 30 min at room temperature. Finally, the slides were developed with AEC+ chromogenic substrate (Dako Cytomation) for 10 min, stained with Lillie-Mayer hematoxylin for 2 min and mounted using aqueous mounting media. 3. Results 3.1. Characterization of monoclonal antibody 7H10 The isotype of Mab 7H10 is IgG1. With 7H10, fluorescence signal was observed in MDCK cells inoculated with H5N1 [(A/turkey/Turkey/1/05), (A/Vietnam/1203PR8/H5N1), (A/ Indonesia/TLL101/H5N1) and (A/Indonesia/CDC594/H5N1)], H5N2 (A/Chicken/Singapore/98) and H5N3 (A/Chicken/Singapore/97), while no significant signal was detected in other non-H5 infected cells [(A/Puerto Rico/8/34/H1N1), (A/Duck/Nanchang/4184/00/H2N9), (A/chicken/Singapore/02/H3N2), (A/shorebird/DE/ 12/04/H6N8) and (A/chicken/Singapore/94/H7N1)] or uninfected cells (Fig. 1A). This result validates that 7H10 can recognize H5 subtype AIV specifically. Also, the same experiment was performed with recombinant baculovirus-infected SF9 cells (Fig. 1A). HA expressed in recombinant baculovirus was cloned from an H5 strain (A/goose/Guangdong/97/H5N1). Fluorescence signal was observed
Table 1 Lack of neutralizing activity of 7H10 to H5 AIVs. Monoclonal antibodies
7H10 5B5 8B6c 3F1d a b c d
A/Vietnam/1203/04 (clade 1)
A/Indonesia/CDC594/06 (clade 2.1)
HIa
VNb
HI
VN
1 1 32 1
10 10 640 10
1 1 16 1
10 10 320 10
HI: Titer in hemagglutination inhibition; titer of 1 indicates a negative result. VN: Titer in virus neutralization with MDCK cells; titer of 10 indicates a negative result. 8B6 is a neutralization antibody to H5, used as a positive control. 3F1 is a non-HA antibody, used as a negative control.
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Fig. 1. Characterization of the monoclonal antibody 7H10. (A) Recognition of HA in H5 AIV-infected MDCK cells and baculovirus expressed rHA1 by 7H10 in immunofluorescence assays. MDCK and SF9 cells were infected with variant subtypes AIV or recombinant baculoviruses, respectively; H5N1*: MDCK cells were infected with H5N1 human strain (A/Indonesia/CDC594/2006); the rest were infected with avian strains or attenuated strains from countries as indicated, which are A/turkey/Turkey/1/05, A/Vietnam/1203PR8/H5N1, A/Indonesia/TLL101/H5N1, A/chicken/Singapore/98/H5N2, A/chicken/Singapore/97/H5N3, A/Puerto Rico/8/34/H1N1, A/Duck/Nanchang/4-184/00/H2N9, A/chicken/Singapore/02/H3N2, A/shorebird/DE/12/04/H6N8 and A/chicken/Singapore/94/H7N1. (B) Western blot. 7H10 can detect HA1 protein of H5N1 virus (A/goose/Guangdong/97 (H5N1)) expressed in E. coli total cell lysate. Negative control is the blotting with RPMI medium.
only in H5-expressing SF9 cells, indicating that 7H10 recognizes the H5 protein, but not any other viral proteins from H5 AIV. Further, in Western blot, recombinant E. coli-expressed H5 HA1 protein (A/goose/Guangdong/97/H5N1) was detected from total cell lysate with 7H10. The protein size shown on the nitrocellulose membrane was 36 kDa (Fig. 1B), which is equivalent to the molecular weight of HA1. This finding reveals that 7H10 reacts against a linear epitope on the HA1 protein of H5. Meanwhile, 7H10 fails to neutralize any of H5 AIVs either in MDCK cells or in chicken embryos. It does not have HI activity to any of H5 AIVs tested (Table 1), confirming that 7H10 does not target any neutralizing epitope. 3.2. Detection of natural infection of H5 subtype virus in formalin-fixed tissues H5N1 AIV-infected tissue samples from Hong Kong, which were collected from the years 2002 to 2006, were tested with 7H10 in immunohistochemical staining. These tissues include different types of organs, such as brain, kidney, liver, lung and pancreas from various avian species, including magpie robin (Copsychus saularis), pond heron (Ardeola bacchus), chicken (Gallus gallus) and grey heron (Ardea cinerea). H5 expression was observed in every infected sample in the staining with 7H10 (Fig. 2). In kidneys, there was a wide distribution of intensive signals throughout the renal tubular cells.
In the lung tissue, the bronchiole lumen showed strong signals at the lining of the epithelial cells. Also, H5 expression was found in individual epithelia. In the brain tissue, the staining signal was observed in cytoplasm of glial cells and neurons. None of samples stained with RPMI medium displayed any specific signal. These data indicate that 7H10 recognizes H5 expression in formalin-fixed tissues. Further, in avian tissue specimens infected with H5 AIV from Indonesia, similar positive results were obtained by 7H10. These samples include different organs, such as thymus, kidney, liver, lung, ovary and pancreas from 2005 to 2006. This underlines that 7H10 can detect the expression of H5 strains, from different regions, in formalin-fixed tissues. 3.3. Detection of experimental infection with variant H5 AIV human isolates in formalin-fixed tissues To investigate further whether the 7H10 is able to detect variant H5 AIVs, especially H5N1 human isolates, 24 different human H5N1 strains from Indonesia and 8 avian strains were collected from Indonesia, Vietnam and Singapore, including 3 non-H5 strains. Experimental infection was performed in chicken embryos and mice with these viruses. Brain tissues from infected chicken embryos were tested with 7H10 to locate H5 antigen expression.
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Fig. 2. Immunohistochemical detection of H5N1 AIV in paraffin-embedded tissue sections of different organs from avian species infected with Hong Kong strains using 7H10. Organs obtained from magpie robin (C. saularis) were preserved in paraffin embedding (brain, lung and kidney) and subjected to IHC staining as described. (−): negative controls stained with RPMI medium. Magnification, ×32.
H5 expression was observed exclusively in cytoplasm of glial cells and neurons from infected chicken embryos (Fig. 3A). Infected cells were extensively distributed through the entire brain tissue. Compared with normal brain tissues, glial cell proliferation was also observed among H5-expressing cells as lesions caused by highly pathogenic AIV infection. Among all, 23 of the 24 human strains and all of these avian H5 strains were successfully detected by 7H10 in IHC staining, while none of those samples infected with non-H5 strains presented any positive staining with 7H10 (Fig. 3C). The antibody targeting nucleoprotein was used to validate AIV infection in these chicken embryos. These results confirm that 7H10 is an H5-
specific monoclonal antibody without cross activity to any other AIV subtypes in IHC staining in formalin-fixed tissues. Mice infected experimentally with human H5N1 isolates were used in this study to test 7H10 activity in mammalian tissues. Among the mouse organs tested, positive signals were mainly observed in lung and brain tissues. In the infected lung tissue, the H5 viral antigen expresses in cytoplasm of the respiratory epithelium and alveolar macrophages (Fig. 4A and B), as well as in cellular debris of the bronchi lumen and bronchioles. In the infected brain tissues, H5 antigen expression was observed in cytoplasm of individual neurons and glial cells, and parts of infected cells became
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Fig. 3. Immunohistochemical detection of H5N1 AIV in paraffin-embedded brain tissue sections of chicken embryos infected experimentally with an Indonesia human strain (A/Indonesia/TLL001/2006/H5N1) using 7H10. (A) Viral antigens were detected in the brain tissue of 10-day-old chicken embryos. Arrow: glial cell proliferation caused by infection; (B) no viral antigen and lesion was detected in the brain tissue of uninfected chicken embryos stained with 7H10 in IHC assays; (C) brain tissues from chicken embryos infected with variant AIV strains were stained with 7H10 and anti-NP antibody in IHC assays. H5N1: A/Indonesia/TLL005/06/H5N1H9N2; H3N2: A/chicken/Singapore/02/H3N2; H7N1: A/chicken/Singapore/94/H7N1; A/chicken/Singapore/98/H9N2. Magnification, ×96.
degenerative (Fig. 4C and D), indicating systemic infection of these highly pathogenic H5N1 strains. This result reflects that 7H10, which is able to detect H5 in formalin-fixed tissue, is effective in mammalian tissues as well as in avian tissues. 3.4. Immunohistochemical detection of H5N1 infection in one human tissue sample with 7H10 A human lung tissue was collected from one patient who died from H5N1 AIV infection in 2006. H5N1 infection in this case was identified by reverse-transcription PCR performed previously in Indonesia. The human lung specimen was fixed in 10% buffered formalin and tested with 7H10 in IHC staining. H5 expression due to H5N1 infection was detected in lung epithelium cells (Fig. 5A). Compared to mouse specimens, H5 signals were sporadic in the human lung tissue. In those infected cells, H5 expression distributed throughout cytoplasm. This result indicates that 7H10based IHC method is sensitive enough to detect H5 infection in human tissues, suggesting that 7H10 can be employed for additional studies on human pathology during H5N1 infection. 3.5. Identification of an H5-specific linear epitope for 7H10 Immunoblotting using 7H10 showed that the epitope was primarily found in the overlapping region of fragment B and C (aa 201–266) from the HA1 of H5 subtype (A/goose/Guangdong/97/ H5N1). To identify the C terminal of this epitope, 8 truncated
fragments were designed and screened with 7H10 (Fig. 6A) in immunoblotting. The amino acid 251 on HA1 was found to be the last amino acid of the epitope for 7H10. To locate the N terminal of this epitope, 8 mutated fragments were designed by primers with mutated sequences and screened with 7H10. Among the 8 mutants, amino acids 240–247 on HA1 were changed to alanine, individually, by specific primers. As shown in Western blot, the first amino acid in the epitope is the amino acid 244 on HA1 (Fig. 6B). This indicates that the linear epitope of 7H10 is exactly located from the amino acid 244 to 251 on the hemagglutinin of H5 subtype, the sequence of which is “Phe Phe Trp Thr Ile Leu Lys Pro” (FFWTILKP). To verify the epitope sequence further, HA gene-sequencing was performed with the 24 human AIV strains. 21 of them carry identical epitope sequences, while two detectable strains (A/Indonesia/CDC329/2006, ABI36050 and A/Indonesia/CDC326/ 2006, ABI36048) have the compatible substitution in one residue on the epitope (FFWTILNP), suggesting the detection range of 7H10 for H5 strains could be even larger. However, for the only strain which can not be detected in our immunohistochemistry test (A/Indonesia/CDC669/2006, ABI36450), the epitope was found to be “FFWAILKP”. The mutation from threonine to alanine corresponds to a mutant designed for epitope mapping, which could not be detected by 7H10 in immunoblotting either. The detection range of 7H10 for H5 subtype AIV was further studied by searching the epitope sequence of 7H10 in the database of AIV genome provided by NCBI influenza resource (Table 2). Up to June of 2007, among 1088 full-length sequences of H5 in the
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Fig. 4. Immunohistochemical detection of Indonesia H5N1 AIV in paraffin-embedded lung and brain tissue sections of mice infected experimentally with an Indonesia human strain (A/Indonesia/TLL011/2006/H5N1) using 7H10. (A), (B) Viral antigen expression in respiratory epithelial cells or alveolar macrophage. (C), (D) Viral antigen expression in neurons and glial cells. (A), (C), magnification: ×32; (B), (D), magnification: ×96.
database, 972 cases contain the identical epitope sequence and 98 cases contain compatible epitope sequence for detection, indicating that 7H10 is able to detect over 98.3% of H5 strains reported. Besides, the detection range of 7H10 reaches 100% for H5 AIV strains
from Hong Kong and Vietnam. 7H10 can detect 93.58% of H5 AIV strains from Indonesia due to 6 cases with the undetectable epitope “FFWAILKP”. Meanwhile, no sequence from any other hemagglutinin subtype was found to contain any detectable epitope for 7H10
Fig. 5. Immunohistochemical detection of H5N1 (A/Indonesia/TLL012/2006/H5N1) infection in paraffin-embedded lung tissue sections of humans. (A) Expression of H5 antigen in respiratory epithelial cells; (B) negative controls stained with RPMI medium. Magnification, ×96.
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Fig. 6. Mapping of the epitope of 7H10. (A) Schematic diagram of the hemagglutinin protein 1, showing the expression constructs of the HA1 fragments and their interaction with 7H10 in Western blot. aa, amino acid. (B) Schematic diagram of the mutated hemagglutinin 1 fragments, showing the constructs for the expression of the different mutations on the HA1 fragments and their interaction with 7H10. “+”: positive by 7H10 in Western blot. “−”: negative by 7H10 in Western blot.
Biosafety Level 3 facilities, there is an urgent need for safe and effective diagnostic methods among developing countries that are plagued from H5N1 AIV. Under normal circumstances, viral virus infectivity in the infected tissues is inactivated by the fixation process, which might remove the lipid envelop and destroy natural conformation of viral hemagglutinin (King, 1991; Koch and Csonka, 1958; Okamoto et al., 2004). Therefore, working on formalin-fixed tissues, the IHC method described in this study provides a stepping stone for regulatory agencies to a
in NCBI database, confirming “FFWTILKP” is an H5-specific and common epitope. 4. Discussion Immunohistochemical detection of H5 infection is important for a better understanding in the pathology of H5 AIV in both birds and humans (Gu et al., 2007; Lee et al., 2005; Uiprasertkul, 2007; Uiprasertkul et al., 2005). Due to the lack of sufficient Table 2 H5-specific linear epitopes of 7H10 among H5 AIVs in database by June 2007. Subtype
Region
Sequences with identical epitope
Sequences with detectable epitope
Total sequences
Detectable range
H5 H5 H5 H5 H5 H1 H3 H6 H7 H9
Any Asia Hong Kong Vietnam Indonesia Any Any Any Any Any
972 753 122 127 88 0 0 0 0 0
1070 784 122 129 102 0 0 0 0 0
1088 800 122 129 109 599 1749 204 238 184
98.35% 98% 100% 100% 93.58% 0 0 0 0 0
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Fig. 7. A new H5 monoclonal antibody was used in H5 detection by IHC staining. Brain tissue sections of chicken embryos infected experimentally with an Indonesia human strain (A/Indonesia/CDC669/H5N1) were stained using the new Mab 5B5 or 7H10. Positive signals were observed in 669-infected cells by 5B5.
safer diagnosis on H5 infected animal tissues (Wootton et al., 2006). The results in this report indicate that 7H10 enables effective detection of H5 antigen to be performed in formalin-fixed tissue sections. This finding, together with its activity in Western blotting and the lack of HI and neutralization activities, indicates that 7H10 recognizes a linear epitope on the HA protein. Since a linear epitope is independent of conformation, recognition by the certain Mab is simply determined by the amino acid sequence of the epitope. By Sequence Polymorphism analysis (Biohealth base), each of residues on 7H10 epitope is identified as the major residue among H5 subtype. Even if some variants do not contain the exact epitope sequence, substitutions with similar amino acids are compatible for the antibody binding. Therefore, more than 98.3% of H5N1 AIV worldwide, could be detected by 7H10 based on the exact or compatible epitope. Since such dominant sequence is not found in any other subtype, the sequence “FFWTILKP” is identified as a common and specific epitope on the H5 subtype hemagglutinins. Among those H5 strains with undetectable epitope sequence, 6 strains derived from H5N1 cases reported in 2006 in Indonesia, which carry the “FFWAILKP” epitope. This phenomenon leads to concerns about the vulnerability of 7H10-based detection due to the possibility of antigenic drifts. Therefore, another anti-H5 monoclonal antibody, termed as 5B5, was generated with the antigen from an Indonesia H5N1 strain (A/Indonesia/CDC669/2006), which cannot be detected by 7H10 (Fig. 7). 5B5 recognizes a linear epitope existing in all Indonesia H5 AIV strains and most of non-Indonesia H5 strains (Table 1). Furthermore, the combination of the two Mabs can detect 100% of H5 AIV strains in Indonesia, Hong Kong and Vietnam. Future work will focus on the updating of this Mab cocktail in H5 AIV diagnosis. In addition, all of H5 strains reported in Genebank in 2007 from Indonesia contain the exact “FFWTILKP” epitope, indicating that this epitope continues to be dominant and conserved in Indonesia at current time. The difference in viral antigen distribution and intensity among different species reflects the difference in pathogenicity of H5N1 virus among various host species. This is mainly caused by the difference in the distribution of sialic acid receptor for AIVs (Szollosy
et al., 1952). As shown in the results, the method with 7H10 is able to detect H5 AIV infection in tissue sections from mammals, including humans, indicating its sensitivity in detection and its potential in promoting the understanding of H5 viral pathology in human infection. In conclusion, the use of 7H10, in diagnostic IHC staining, presents three advantages over the current methodologies. Firstly, working on formalin-fixed tissue, 7H10 provides a safe diagnostic approach in H5N1-AIV studies. This approach realizes safer detection of H5 AIV in suspicious samples as they will neither infect humans nor contaminate the environment. Secondly, 7H10 could identify the highly pathogenic H5 subtype AIV efficiently and specifically, which is effective in almost all of H5 influenza viruses in the database without cross activity to other subtypes. This could mean a breakthrough in the AIV diagnosis in the light of its specific detection of H5 viral antigen in formalin-fixed tissue sections. Lastly, the ability of 7H10 to accurately locate H5 viral antigen in infected tissue, facilitates microbiologists and pathologists to obtain information about the stage and pathology of AIV infection in tissue samples in the studies on H5 AIV. Acknowledgements We thank the National Institute of Health, Research and Development, Indonesia, Hong Kong Agriculture and Fisheries Department Tai Lung Veterinary Lab and Institute of Pertanian in Bogor, Indonesia, for contributing H5N1 infected specimens or clinical samples, Ms. Yu Zhu for tissue sectioning, Ms. Huiting Ho for providing virus samples by reverse genetics and Mr. Hongliang Qian for providing control Mabs. References Apisarnthanarak, A., Warren, D.K., Fraser, V.J., 2007. Issues relevant to the adoption and modification of hospital infection-control recommendations for avian influenza (H5N1 infection) in developing countries. Clin. Infect. Dis. 45, 1338–1342. Babakir-Mina, M., Balestra, E., Perno, C.F., Aquaro, S., 2007. Influenza virus A (H5N1): a pandemic risk? New Microbiol. 30, 65–78.
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